A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on serine residues. Their two-dimensional phosphopeptide maps show multiple phosphorlation sites and a considerable similarity to the phosphopeptide maps of lamins labeled in vivo. Photoaffinity labeling experiments revealed several polypeptide fractions in the nuclear lamina fraction that are candidates for the protein kinase(s).The nuclear lamina (NL) is a proteinaceous meshwork located at the inner surface of the nuclear membrane (1, 2). The major constituents of the NL are three polypeptides with molecular masses between 60 and 75 kDa, termed lamins A, B, and C (2). It is thought that the NL plays a role in maintaining the structural organization of both the nuclear envelope (3, 4) and chromatin (5, 6) in the interphase nucleus. It has also been shown that the lamins are members of the intermediate filament protein family (7-11).The nuclear lamins are phosphoproteins. During interphase they exhibit a relatively low level of phosphorylation of about 0.2 mol of phosphate per mol of protein (12). However, when the NL is disassembled in mitosis (3, 4) there is an increased phosphorylation of the three lamins (4, 12). Little is known about the nature, location, and regulation of the protein kinase(s) involved in both interphase and mitotic phosphorylation of the lamins.We have recently developed a method for the isolation of the NL from Ehrlich ascites tumor (EAT) cells (13,14 (Amersham) at 25 uCi/ml in DME otherwise free of phosphate or methionine, respectively, supplemented with 5% dialyzed fetal calf serum.The original method developed for the isolation of NL from EAT cells (13, 14) was modified as follows. One milliliter of packed cells was resuspended in 30 tnl of solution 1 (0.25 M sucrose/5 mM EDTA/5 mM Tris HCI, pH 7.5/0.5 mM phenylmethylsulfonyl fluoride), incubated at 0C for 5 min, and centrifuged at 600 x g for 5 min. The cells were treated once more with 30 ml of solution I and twice with 30 ml of solution 11 (0.25 M sucrose/0.1 mM EDTA/5 mM Tris-HCI, pH 7.5/0.5 mM phenylmethylsulfonyl fluoride). Each time the cells were resuspended (Vortex mixer), incubated for 5 min at 0C, and collected by centrifugation at 600 x g for 5 min. The pellet was then resuspended in 10 ml of solution II, mixed with 10 ml of aqueous 1% Nonidet P-40 (Sigma), layered over a 60-ml cushion of solution III (0.25 M sucrose/0.1 mM EDTA/5 mM Tris HCl, pH 7.0), and spun at 3500 x g for 15 min. The resulting semitransparent pellet was resuspended in 10 ml of solution III and incubated for 30 min at room temperature in the presence of DNa...

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Using a membrane filter retention technique we have studied the interaction between DNA and lysine rich histone H5 in vitro. It is found that, depending on the ionic conditions, H5 can bind DNA in a random or cooperative manner and exhibits a preference to DNA with high molecular weight and/or high A + T content, as also observed with H1. The presence of 6 M urea in the assay mixture does not impair the selectivity of H5 to A + T rich DNA but partly affects its selectivity to DNA size. In contrast to H1, H5 does not discriminate between the superhelical and relaxed forms of circular SV40 DNA.